Treatment and diagnosis of dementia

ABSTRACT

The administration of Somatostatin and growth hormone releasing factor at useful dosage levels alleviates the symptoms of various neuropsychiatric disorders. 
     Growth hormone and neuropsychiatric responses to the administration of growth hormone releasing factor to mammals indicate its usefulness as an antemortem marker for senile dementia of the Alzheimer&#39;s type.

The invention deals with methods for treating somatotropinergicdeficiencies via the use of SS and/or GRF. Thus, pharmaceuticalcompositions containing one or more of SS and GRF are administered viavarious routes to effect improvements in various physiological andpsychological disorders.

The invention also deals with the use of GRF as a marker for SDAT.

BACKGROUND

The somatotropinergic system (STS) is the only neuroendocrine axis inwhich specific stimulatory and inhibitory neuropeptidergic regulatorshave been demonstrated thus far. In normal conditions, growthhormone-releasing factor (GRF) and somatostatin (SS) are thehypothalamic hypophysiotropic hormones responsible for the regulation ofgrowth hormone (GH) secretion.

At the same time, GRF and SS are influenced by central mono-aminergicand peptidergic neuromodulators to optimize the functioning of the STS.See Cacabelos R., Niigawa H., Hariguchi S. "Hypothalamohypophysealsystem and brain function." J. Clin. Sci. 22:1108-1120 (1986).

Recent investigations suggest that the functional structure of the STScharacteristically represented at the peripheral level might exist inthe central nervous system (CNS). Furthermore, SS levels are reduced inspecific areas of the CNS in patients with senile dementia and areelevated in the neostriatum of patients with Huntington's chorea. SeeCacabelos R., Niigawa H., Ikemura Y."Neuroendocrine correlates in seniledementia of the Alzheimer type. "Progr. Clin. Neurosci. 2:231-247(1986); Beal M. F., Uhl G., Mazurek M. F., Kowall N., Martin J. B."Somatostatin: Alterations in the central nervous system in neurologicaldiseases." in: Martin J. B., Barchas J. D., Ed. Neuropeptides inneurologic and psychiatric disease. pp. 215-257 (Raven Press, N.Y.,1986); Epelbaum J. "Somatostatin in the central nervous system:physiology and pathological modifications." Progr. Neurobiol. 27:63-100(1986). In addition, an abnormal rise of GH in response to GnRH or TRHhas been described in several neuropsychiatric disorders. See in BrownG. M., Koslow S. M., Reichlin S. Ed. Neuroendocrinology and psychiatricdisorder (Raven Press, N.Y., 1984). It has been speculated that thecentral regulators of the STS (e.g., GRF, SS) influence higheractivities of the CNS. Such speculation led to search for potentialtherapeutic uses of these neuropeptides in those neurological diseasesin which the normal functioning of the STS is impaired.

The neurochemical characteristics of early and late onset seniledementia of the Alzheimer type (SDAT) can be clearly demonstrated inpostmortem studies in which it is possible to observe that cholinergicand somatostatinergic deficits are more pronounced in patients with anearly onset of the disease. See Rossor M. N., Iversen L. L., Reynolds G.P., Mountjoy C. Q., Roth M. "Neurochemical characteristics of early andlate onset types of Alzheimer's disease." Br. Med. J. 288:961-964(1984). In advanced stages, clinical assessments show that the course ofthe disease is more rapid and prominent in younger patients.

Since dementia may be attributed to various etiologies of which SDATaccounts for at least 50% of the cases, the search for antemortemmarkers capable of establishing an early differential diagnosis foridentifying potentially reversible or treatable causes of dementia isbecoming an extremely important matter.

SDAT is believed to be a multisystem disorder. See Price D. L., StrubleR. G., Whitehouse P. J., Kitt C. A., Cork L. C., Walker L. C., CasanovaM. F. "Alzheimer's disease: A multisystem disorder. "in: Martin J. B.,Barchas J. D., (Ed). Neuropeptides in neurologic and psychiatricdisease. pp. 209-214 (Raven Press, N.Y., 1986). The most relevantpeptidergic abnormalities in SDAT involve a clear deficit in corticalsomatostatin and corticotropin-releasing factor (CRF). Other peptidergicand monoaminergic systems are also affected.

THE INVENTION

It has been discovered that growth hormone releasing factor (GRF) andsomatostatin (SS) when administered together or separately, can treatneuropsychiatric disorders in some mammals. Specifically either or bothexert significant effects on locomotor activity and improve learningabilities. It has also been found that the GRF-induced GH response canbe taken as a useful marker for the early stages of SDAT.

PEPTIDES

Various commercially available forms of SS and GRF can be employed inthe invention. Biologically active fragments can be substituted for allor part of the SS or GRF used. Somatostatin is generally characterizedas growth hormone-releasing inhibiting factor. It is compound No.8561 inMerck Index, 10th ed. (1983). It is preferred that the 1-14 fragment(i.e., SS-14) be employed herein. Various commercial products containinguseful forms of SS may be used herein. Mixtures are operable.

Growth hormone releasing factor is chemically describable as thestimulatory growth-hormone releasing factor of the hypothalamus thatassists in the nueroregulation of growth hormone secretion. It iscompound No.4416 in Merck Index, 10th ed. (1983). Preferred for useherein are the segments or fragments designated as 1-44 and 1-29.Various commercial products containing useful forms may be used herein.Mixtures are operable.

DOSAGES

Pharmaceutical preparations used in accordance with the invention willcontain one or more active substances including at least one of SS andGRF along with the optional inclusion of pharmaceutically acceptablecarrier(s). Peptide levels of about 1 to about 10 mcg/kg body weighti.v. and of about 5 to about 50 mcg/kg body weight s.c. are operable.The length of time for which the SS and/or GRF-containing composition isadministered can vary greatly. Depending upon the effect desired, dailyadministration may be preferred. Administration every 6 or 12 hours canalso be used. Useful times of administration depend upon the preferencesof the treating physician.

IN THE DRAWINGS:

FIG. 1

Computer recordings of behavioral patterns of rats treated withsomatostatin and growth hormone-releasing factor in a maze paradigm.

A. Behavioral pattern of prototype control rat (5 mcg 0.9% saline,i.c.v.) in a maze paradigm.

B. Behavioral pattern of a rat treated with somatostatin (SS-14; 1 mcg,i.c.v.).

C. Behavioral pattern of a rat treated with growth hormone-releasingfactor (GRF₁₋₄₄ ; 1 mcg, i.c.v.).

D. Behavioral pattern of a rat treated with GRF₁₋₄₄ (5 mcg, i.c.v.).

The animals had to learn to escape from the shock area in order to avoida continuous 1.5 mA electric shock. Experimental time --30 minutes.

FIG. 2(A-F)

Effects of somatostatin and GRF on locomotor activity and learning in amaze paradigm.

The rats were treated intracerebroventricularly with SS-14 (1 mcg) andGRF₁₋₄₄ (1 mcg) and their behavior was automatically recorded bycomputer using the maze paradigm shown in FIG. 1. p/t denotes pulses pertime unit (minute). Locomotor activity was quantified in pulses perminute or cm² /min. Total inputs (I/O) in neutral position (white bars)and shock position (black bars) denote how many times the animals enterneutral and shock areas. The N/S ratio results from dividing I/O inneutral position by I/O in shock position and is interpreted as an indexof learning in the 30-minute experimental period. The positional time(seconds) represents how long the animals stay in neutral (white bars)or shock (black bars) areas. N-sp and S-sp (in seconds) indicate thetime-latency per movement or input in neutral (white bars) and shock(black bars) areas, respectively.

p<0.005 vs C (control)

p<0.005 (NA vs SA)

FIG. 3

It shows GRF-induced GH response in elderly subjects ( ) and patientswith early ( ) and late onset senile dementia of the Alzheimer type ( ).

* p<0.005 vs. basal level (0) (means ±SD)

** p<0.005 vs. control ( ).

FIG. 4

It illustrates the correlation between the GRF-induced GH response 60minutes after injection and the mental performance of patients withearly onset senile dementia of the Alzheimer type 24 hours prior totesting. The following Examples illustrate the invention.

EXAMPLE 1

To clarify whether SS and GRF display at the central level the sameantagonistic effect that they have at the pituitary level, the effectsof these neuropeptides on several behavioral parameters have beenstudied using the OUCEM-86^(TM) (Osaka University ComputerizedElectronic Maze). The system is integrated by the following components:a Programmable Electronic Platform (PEP) (620×620 mm) equipped with 48photo beam sensors (OPX-T30); a Programming Panel (PP) (Model BECM-0064)for paradigm stimulation; a Shock Generator Scrambler (SGS) (ModelBSG-1065) to apply continuous or discontinuous electric current to thePEP; a Control Station (CS) (BEC 16-Bio Computer Control StationBCS-1105; Bio Medica, Ltd., Osaka) which processes inputs/outputs as aninterface device to automatically computerize behavioral parameters; anda Computer System (NEC PC-9801 VM2) with 1 megabyte Random Access Memory(RAM) for data processing and experimental setting.

Male Wistar rats (175-200 g) (N =5-8 rats/group) receivedintracerebroventricular (i.c.v.) injections of GRF₁₋₄₄ and SS-14 over arange of 0.1 to 10 mcg. Then the rats were studied in the OUCEM undertwo different paradigms (e.g., open field and maze paradigms).

The animals were placed in the PEP for 30-minute periods and locomotoractivity (LA) was automatically recorded in an open field paradigm.Total LA is represented as total inputs (I/O) including movements infour-footed position (4F) and in two-footed position (2F), grooming,rearing and jumping activity, while linear (horizontal) movements (4F)are represented as pulses per minute (pm) or cm² /min

The i.c.v. administration of SS (1 mcg) provoked quantitative andqualitative changes with a significant decrease in LA (31.16±6.90 pm vs20.88±2.82 pm, t=3.08, p<0.02) while GRF (1 mcg) induced a hyperkineticsyndrome characterized by compulsive, uncontrolled movements with an LArate of 47.60±5.35 pm (t=4.21, p<0.005)(control =1122±248 cm² /min; GRF=1713.6±69.5 cm² /min., t =18.73, p<0.005). These changes weredose-dependent (SS:ED₅₀ =1.83 nmol, E_(max) =6.10 nmol; GRF₁₋₄₄ ED₅₀=99.1 pmol, E_(max) =1.98 nmol; GRF1-29:ED₅₀ =297 pmol, E_(max) =5.95nmol).

EXAMPLE 2

This example shows the effects of the intracerebroventricularadministration of SS and GRF on locomotor activity and learning in amaze paradigm.

Using the maze paradigm shown in FIG. 1 in which the rats must learn toescape from the shock area in order to avoid a continuous 1.5 mAelectric shock, SS (1 mcg, i.c.v.) significantly reduced LA 17.12±5.42vs 6.01±1.45 pm, t=4.42, p<0.005) FIGS. 1B,3) and GRF increased LA in adose-related manner (26.66±3.15 pm, t=3.40, p<0.01 at 1 mcg, FIG. 1C;and 90.2±8.4 pm, t=17.51, p<0.005, at 5 mcg, FIG. 1D). The amount ofinputs (I/O) induced by SS was 103.5±29.5 (control =216.25±55.19,t=8.49, p<0.005) in the neutral area (NA) and 77.14±13.5 I/O in theshock area (SA) (control =298±40, t=1.26, n.s.). GRF generated459.35±32.5 I/O in NA (t=18.13, p<0.005) and 340.5±63.5 I/O in SA(t=9.07, p<0.005) (FIGS. 1C,3). The NA/SA (N/S) ratios for control, SS-and GRF-treated rats were 0.72, 1.33, and 1.35, respectively (FIG. 2).

Our results clearly show that GRF and SS exert antagonistic effects onLA in novelty conditions in both open field and maze paradigms (FIGS.1,2). If we consider the N/S ratio as in index of learning, it seemsthat both SS and GRF improve learning abilities at doses ofapproximately 6.10×10⁻¹⁰ mol and 1.98×10⁻¹⁰ mol, respectively.

However, since GRF induced a hyperkinetic syndrome in which the I/O rateas well as the positional time in SA were significantly higher than inthe control group, it could be inferred that SS is more effective thanGRF in improving learning. Indeed, the GRF-induced locomotor agitation(hyperkinetic syndrome) constitutes a serious drawback for the animaltrying to differentiate and discriminate patterns in the maze (FIGS.1C,D).

EXAMPLE 3

Twenty female in-patients with primary degenerative dementia (Alzheimerlike) aged 67-84 years were divided in two therapy-groups with GRF(1-29) NH₂. All patients were treated with this product for 8 days witheither of the following schedules:

(I) 250 mcg s.c. twice daily

(II) 50 mcg i.v. twice daily

Therapy was started after a 15-day wash-out period forpsychotropic-drugs.

Evolution of patients was assessed using the MMS Folstein test andChrichton's scale test at 12 hours, 24 hours and 7 days after startingwith GRF. The statistical evaluation was made using Friedman test with arisk level p<0,01 (highly significant differences).

Both groups show a significant increase in both scores and clinicalfindings, more important in the group with i.v. GRF administration thanin the one with s.c. administration. The effect starts at 12 hours afteradministration and remains significant over the whole 7 days oftreatment.

The lower scores in the Crichton's scale observed for the I group areprobably related with the fact that, since inclusion in either group wasdependent on the difficulty of the i.v. puncture, the patients includedin the s.c. group were those whose clinical situation was worst.

During the treatment with GRF the nurse in charge of the patientsreported an improvement of the patient's situation regarding sphinctercontrol and general psychomotricity. This improvement returned topretreatment situation when the GRF administration was discontinued.

After finishing GRF administration the same groups have been evaluatedwith MMS-Folstein test and Crichton's scale test in order to both assessthe test's reproductibility and to establish the elimination of the GRFeffect thus confirming the significant effect of GRF (1-29) NH₂ in thosepatients. The variation coefficients were 12% for MMS-Folstein test andthe 2.7% for Chrichton's scale test.

EXAMPLE 4

The subjects were divided into three groups:

(a) controls (N =9): 5 females and 4 males (age =70.10±2.76 years; range=66-75);

(b) inpatients with early onset senile dementia of the Alzheimer type(EOSDAT) (N =10): 5 females and 5 males (age 65.00±3.31 years; range=57-69); and

(c) inpatients with late onset senile dementia of the Alzheimer type(LOSDAT) (N =10): 5 females and 5 males (age =75.70±3.77 years; range=70-82).

All the SDAT patients met the DSM-III criteria for primary degenerativedementia and rigorous diagnostic criteria for SDAT. As EOSDAT patientswere considered those whose insidious memory loss or deterioration ofcognitive functions began before the age of 60 and had a progressivecourse of more than 2 years. In LOSDAT, the onset of the diseaseoccurred after the age of 60. The degree of severity of the dementia wasassessed with Hasegawa's Dementia Rating Scale (DRS) (Hasegawa K., InoueK. "An investigation of dementia rating scale for the elderly."Seishinigaku 16:965-969, 1974), Folstein's Mini-Mental State (MMS) Test(Folstein M. F., Folstein S., McHugh P. "Mini-mental state: a practicalmethod for grading the cognitive state of patients for the clinician."J. Psychiat. Res. 12:189-198, 1975) and a modified model of the BriefCognitive Rating Scale (BCRS) and Functional Assessment Stages (FAST) ofReisberg (Reisberg B., Ferris S. H., De Leon M. J. "Senile dementia ofthe Alzheimer type: Diagnosis and differential diagnostic features withspecial reference to functional assessment staging." in: Traber J.,Gispen W. H. (Ed). Senile dementia of the Alzheimer type. pp 18-37,Springer-Verlag, Berlin, 1985). Concomitant pathology other than SDATwas discarded under a careful screening of the subjects with CT Scan,EEG, ECG and complementary laboratory data. Current diagnosis or historyof SDM-III major affective disorder or schizophrenia as well as historyof endocrine disease were criteria for exclusion. All of the controlsubjects were free of psychiatric and endocrine illness. All subjectsincluded had been drug-free for a minimum of 7 days before testing.

A 21-gauge indwelling venous needle with an attached three-way stopcookwas inserted into the antecubital vein. Baseline blood samples for GHdetermination were collected immediately before testing. GRF(1-44)NH₂was then injected as an intravenous bolus (100 mcg), and blood sampleswere obtained at 15, 30, 45, 60, 90 and 120 minutes after injection fordetermination of GH concentrations in plasma. The plasma GH levels weremeasured by radioimmunoassay using a commercial GH-RIA-Kit (Dainabot Co.Ltd. Tokyo). The intra- and inter-assay coefficients of variation were6.9% and 10.6%, respectively.

Simultaneously to the performance of the GRF test,electroencephalograhic and cardiovascular monitorization of the subjectswere carried out. Twenty-four hours prior to the GRF test, and 3, 12, 24and 48 hours after testing, the DRS, MMS, BCRS, and FAST tests wereperformed to evaluate mental performance and behavior. Additionalmonitoring of GRF effects was done by global physician assessment andevaluation of the Mihara Nursing Scale (MNS). This test was developed tobe used as a non-cognitive parametric indicator involving several items,including quantification of food intake, locomotion, social interaction,and complementary laboratory data.

The results were analyzed statistically by the Student's test, analysisof variance (ANOVA) and the Mann-Whitney test using a NEC PC-9801VM2computer.

GRF induced a marked increase in the levels of plasma GH in from 30 to90 minutes after injection with a maximum peak (15.61±5.71 ng/ml, t=6.08, p <0.005) at 60 minutes in EOSDAT (FIG. 3). This response wasabsent in both LOSDAT and control subjects, in whom plasma GH increasedslightly but never reached a significant level over basal concentrations(FIG. 3).

In the EOSDAT responders, the rise of plasma GH levels after GRFinjection showed a great individual variability (range =5.5-28.75ng/ml). This response was higher in females (19.74 ±4.86 ng.ml) than inmales (11.52±2.82 ng/ml, p<0.005) (see the Table). No sexual differenceswere evident in the other two groups. Significant differences in thebasal plasma GH concentrations among the three groups were not detected.

                                      TABLE 1                                     __________________________________________________________________________    SEX- AND TIME-DEPENDENT RESPONSES OF GROWTH HORMONE TO GROWTH                 HORMONE-RELEASING FACTOR IN PATIENTS WITH SENILE DEMENTIA AND                 ELDERLY SUBJECTS                                                              TIME CONTROL                                                                              CONTROL                                                                              EOSDAT EOSDAT  LOSDAT LOSDAT                               (min.)                                                                             (females)                                                                            (males)                                                                              (females)                                                                            (males) (females)                                                                            (males)                              __________________________________________________________________________    0    1.03 ± 0.94                                                                       1.07 ± 0.92                                                                       1.92 ± 1.52                                                                       1.99 ± 1.67                                                                        1.09 ± 1.01                                                                       0.95 ± 0.98                       15   3.09 ± 1.58                                                                       2.21 ± 1.87                                                                       2.23 ± 1.90                                                                       2.70 ± 1.92                                                                        2.04 ± 1.01                                                                       2.13 ± 0.94                       30   3.68 ± 2.09                                                                       3.16 ± 2.76                                                                       6.26 ± 2.05                                                                       5.49 ± 1.91                                                                        3.65 ± 2.35                                                                       2.81 ± 1.90                       45   4.28 ± 3.38                                                                       5.17 ± 3.46                                                                       14.86 ± 3.57                                                                       6.87 ± 2.84*                                                                      4.68 ± 3.18                                                                       3.61 ± 3.51                       60   2.97 ± 1.96                                                                       3.17 ± 2.61                                                                       19.74 ± 4.86                                                                       11.52 ± 2.82**                                                                    4.97 ± 3.86                                                                       4.09 ± 2.56                       90   2.04 ± 1.38                                                                       1.88 ± 1.87                                                                       11.28 ± 5.72                                                                      7.41 + 2.79                                                                           4.04 ± 3.22                                                                       3.08 ± 2.22                       120  1.10 ± 0.95                                                                       0.91 ± 0.87                                                                       5.09 ± 3.01                                                                       2.73 ± 1.71                                                                        2.44 ± 2.05                                                                       1.64 ± 0.82                       N    5      4      5      5       5      5                                    AGE  70.20 ± 2.78                                                                      70.00 ± 2.73                                                                      65.00 ± 4.25                                                                      65.00 ± 2.00                                                                       76.20 ± 4.48                                                                      75.20 ± 2.00                      RANGE                                                                              67-75  66-73  57-69  62-68   70-82  73-80                                STAGE                                                                               I-II   I-II  VI-VII V-VII   VI-VII VI-VII                               __________________________________________________________________________     EOSDAT = Early Onset Senile Dementia of the Alzheimer Type                    LOSDAT = Late Onset Senile Dementia of the Alzheimer Type                     *p < 0.01 vs. females                                                         **p < 0.005 vs. females                                                  

EEG was abnomal in 95% of SDAT patients due to the advanced stage of thedisease. Basic electroencephalographic patterns for controls (N =6) andSDAT subjects (N =7, prior to the GRF test were 9.27±0.11 Hz/47.36±3.36uV and 8.08±0.67 Hz/40.80±6.06 uV, respectively. Global EEG evaluationafter testing yielded a basic rhythm of 9.05±0.12 Hz(p<0.025)/49.67±5.75 uV for controls and 7.46±0.53 Hz/43.69±5.12 uV forSDAT. Sequential EEG analysis revealed a fall in frequency (8.75±0.25Hz, p<0.005) and an increase in amplitude (55.60±6.40 uV, p<0.02) incontrol subjects and in SDAT patients (7.05±0.90 Hz, p<0.05/52.5±9.89uV, p<0.005) in from 15 to 45 minutes after injection, preceding themaximum plasma GH peak.

There was a good correlation of scores between the DRS and MMS tests.Basal scores for DRS/MMS prior to the GRF test were 3.5±1.75/3.5±2.65.After testing, scores increased to 5.5±2.10/5.15±3.75 (3 hr),6.75±3.25/5.5±3.65 (12 hr), 4.35±1.95/4.5±2.75 (24 hr), and3.5±1.55/4.35±2.75 (48 hr).

Although these differences were not statistically significant, they wereclinically relevant. An inverse curvilinear relationship (Fig.4) betweenthe variables mental performance (x) and maximum GH response to GRF at60 minutes (y) was observed (r =-0.83). A careful evaluation of the BCRSshowed that the axes more significantly affected by GRF were Axis 4(orientation, 30%), Axis 7 (psychomotor, 50%), and Axis 8 (mood andbehavior, 40%). According to the MNS, appetite increased in 45% of thepatients and in 30% of the controls (2 patients developed a bulimicsyndrome), and social interaction improved in 40% of the patients. Allthese behaviors were transient.

These results demonstrate that GRF induces a significant increase of theplasma GH levels 60 minutes after injection in EOSDAT. This GH responseis about 15 minutes delayed with respect to the small peak observed incontrol subjects. Furhtermore, the GRF-induced GH response slightlycorrelates with the severity of the disease and is accompanied by EEGand behavioral changes mainly circumscribed to psychomotor functions.

Reasonable variations, such as those which would occur to a skilledartisan, can be made herein without departing from the scope of theinvention.

What is claimed is:
 1. A method of detecting early onset senile dementiaof the Alzheimer type in a subject comprising administering to thatsubject an effective amount of growth hormone releasing factor andmonitoring the growth hormone response thereto, whereby an elevatedresponse versus normal controls is indicative of early onset seniledementia of the Alzheimer type.
 2. The method of claim 1 whereinabout 1to about 10 mcg/kg (i.v.) or about 5 to about 50 mcg/kg (s.c.) of growthhormone releasing factor.
 3. The method of claim 2, wherein the growthhormone releasing factor is GRF₁₋₂₉.
 4. The method of claim 2, whereinthe growth hormone releasing factor is GRF₁₋₄₄.